Journal of Experimental Botany, Vol. 68, No. 5 pp. 1109–1122, 2017
doi:10.1093/jxb/erw493 Advance Access publication 15 February 2017
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER
A (–)-kolavenyl diphosphate synthase catalyzes the first step
of salvinorin A biosynthesis in Salvia divinorum
Xiaoyue Chen1, Anna Berim1, Franck E. Dayan2 and David R. Gang1,*
1 2 Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA
Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO 80523–1177, USA
* Correspondence: [email protected]
Received 5 August 2016; Editorial decision 13 December 2016; Accepted 16 December 2016
Editor: Qiao Zhao, Tsinghua University
Abstract
Salvia divinorum (Lamiaceae) is an annual herb used by indigenous cultures of Mexico for medicinal and ritual purposes. The biosynthesis of salvinorin A, its major bioactive neo-clerodane diterpenoid, remains virtually unknown.
This investigation aimed to identify the enzyme that catalyzes the first reaction of salvinorin A biosynthesis, the
formation of (–)-kolavenyl diphosphate [(–)-KPP], which is subsequently dephosphorylated to afford (–)-kolavenol.
Peltate glandular trichomes were identified as the major and perhaps exclusive site of salvinorin accumulation in
S. divinorum. The trichome-specific transcriptome was used to identify candidate diterpene synthases (diTPSs). In
vitro and in planta characterization of a class II diTPS designated as SdKPS confirmed its activity as (–)-KPP synthase
and its involvement in salvinorin A biosynthesis. Mutation of a phenylalanine into histidine in the active site of SdKPS
completely converts the product from (–)-KPP into ent-copalyl diphosphate. Structural elements were identified that
mediate the natural formation of the neo-clerodane backbone by this enzyme and suggest how SdKPS and other
diTPSs may have evolved from ent-copalyl diphosphate synthase.
Key words: class II diterpene synthase, diterpenoid diversification, (–)-kolavenyl diphosphate, (–)-kolavenol, neo-functionalization,
neo-clerodane diterpenoid, product specificity, repeated evolution, Salvia divinorum, salvinorin A biosynthesis.
Introduction
Salvia divinorum (Lamiaceae) is a powerful hallucinogenic plant
traditionally used in psycho-spiritual and healing ceremonies
by the Mazatecs of Oaxaca in southern Mexico (Wasson, 1962).
Its psychoactivity is largely due to salvinorin A, a highly selective kappa-opioid receptor agonist (Roth et al., 2002; Butelman
and Kreek, 2015), whose unique structure and distinct pharmacological features make it a valuable template/lead for structure–function explorations (Tejeda et al., 2012; Butelman and
Kreek, 2015; Simonson et al., 2015). Other related metabolites
from S. divinorum such as (–)-kolavenol, hardwickiic acid, and
salvinorin B also exhibit strong biological activities (Pittaluga
et al., 2013; Marchant et al., 2016). Salvia divinorum is the only
known natural source of salvinorin A. While total chemical
synthesis of salvinorin A is possible (Nozawa et al., 2008), it is
not commercially feasible. Elucidation of the natural biosynthetic pathways leading to salvinorin A will facilitate its production and structural diversification for research and medical
applications through synthetic biology approaches.
© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
1110 | Chen et al.
Structurally, salvinorin A is a neo-clerodane diterpenoid, belonging to the superfamily of labdane-related diterpenoids, which currently accounts for over 7000 distinct
chemical entities (Peters, 2010; Zi et al., 2014). Further subdivision of clerodane diterpenoids into neo- and ent-neoclerodanes is based on the stereochemical configurations
of their hydrocarbon backbones (Tokoroyama, 2000). The
biosynthesis of diterpenoids in plants begins in plastids
with the formation of their common precursor, geranylgeranyl diphosphate (GGPP) via the deoxyxylulose phosphate
pathway. In angiosperms, labdane-related diterpenoids
are then typically produced in two reaction steps by pairs
of often plastidial class II and class I diterpene synthases
(diTPSs) (Peters, 2010). The first step, the protonation-initiated cyclization of GGPP, is mediated by a class II diTPS
through formation of the common bicyclized labda-13-en8-yl+ diphosphate intermediate (Fig. 1A) and its conversion into bicyclic prenyl diphosphates (Peters, 2010). The
structural diversity of hydrocarbon skeletons generated by
class II-catalyzed cyclization reactions largely depends on
the absolute configuration of this intermediate and how
it is further processed. For instance, deprotonation of the
Fig. 1. Relevant reactions and proposed salvinorin A biosynthesis pathway. (A) The scheme of reactions catalyzed in routes a and b using GGPP as
the substrate with formation of intermediate labda-13-en-8-yl+ diphosphate. (B) The proposed biosynthetic pathway of salvinorin A based on known
compounds identified from S. divinorum. The step elucidated in this study is highlighted in the box to the lower left. The compounds outlined in the
dashed box could exist as the corresponding descarboxylmethylated molecules and then be converted to their methylesters just prior to formation and
secretion. Alternatively, the carboxylmethyltransferase could act early in the pathway.
A diterpenoid synthase involved in salvinorin A biosynthesis | 1111
methyl group at C-8 of the intermediate leads to formation
of copalyl diphosphate (CPP) isomers, with ent-CPP (9R,
10R, Fig. 1A) and ‘normal’ CPP (9S, 10S) being the most
frequently observed reaction products (Xu et al., 2007a;
Keeling et al., 2010; Brückner et al., 2014; Pateraki et al.,
2014; Cui et al., 2015). If the carbocation is captured by
water prior to deprotonation, labda-13-en-8-ol diphosphate
is formed instead (Schalk et al., 2012; Zerbe et al., 2013;
Pateraki et al., 2014; Cui et al., 2015; Andersen-Ranberg
et al., 2016). Notably, labda-13-en-8-yl+ diphosphate can
also be rearranged through a series of 1,2-hydride and
methyl shifts to give rise to a diphosphate with the clerodane
backbone (Fig. 1A, route a) (Peters, 2010). Products of the
class II diTPSs are substrates for class I diTPSs, which cleave
the diphosphate group, usually followed by cationic cyclization and/or rearrangement reactions. The biosynthesis of
the diverse labdane-related diterpenoids in higher plants is
thought to have evolved from ancestral gibberellin biosynthesis, which involves cyclization of GGPP to ent-CPP by
a class II diTPS, ent-copalyl diphosphate synthase (CPS),
followed by the formation of ent-kaurene by a class I diTPS
(Zi et al., 2014).
Based on the structures of diterpenoids isolated from
S. divinorum (Valdes et al., 1984; Valdés et al., 2001; Bigham
et al., 2003; Munro and Rizzacasa, 2003; Kutrzeba et al.,
2009a), we have outlined a proposed biosynthetic route to
salvinorin A (Kutrzeba et al., 2007, 2009b) (Fig. 1B). The
backbone of salvinorin A and the common modular mechanism employed in the biosynthesis of labdane-related
diterpenoids allowed us to hypothesize that salvinorin
A biosynthesis involves a class II diTPS catalyzing the conversion of GGPP into (–)-kolavenyl diphosphate [(–)-KPP],
which is further dephosphorylated by a class I diTPS into
(–)-kolavenol, the first isolated putative pathway intermediate. Subsequent modifications yield salvinorin A and are
accompanied by the accumulation of an array of related
diterpenoids representing pathway intermediates and minor
products (Fig. 1B). This current investigation identified and
characterized the (–)-KPP synthase (SdKPS) involved in
salvinorin A biosynthesis.
Materials and Methods
Chemical sources
GGPP was obtained from Echelon (Salt Lake City, UT, USA).
(–)-Kolavenol and hardwickiic acid were purchased from BioBioPha
(Kunming, Yunnan, China). Salvinorin B and salvinorin A were
from Sigma-Aldrich. Apigenin was from Indofine.
Plant material growth and culture
S. divinorum (line ‘Tucson’) plants were clonally propagated from
stem cuttings placed in glass flasks filled with distilled water and
transplanted to soil (Frog Smart Naturals potting soil, Foxfarm Soil
& Fertilizer Co., Arcata, CA, USA) after 4 weeks of root formation. Plants (during and after rooting) were grown under controlled
conditions with a 16/8 h light/dark cycle, temperatures of 24 /18 °C,
and humidity kept at 20–50% in a growth room. Fluorescent lamps
(Spectralux) produced light intensities between 100 PAR (plant
bases) and 450 PAR (plant tops).
Metabolite extracts
Salvia divinorum tissue samples and leaf disc samples of Nicotiana
benthamiana were ground under liquid nitrogen with a mortar and
pestle. A portion of frozen powder was used for RNA extraction for
transcript profiling of S. divinorum leaves at different stages. Around
200–250 mg of frozen powder was weighed and suspended in 2 ml
of a methyl tert-butyl ether (MTBE):ethyl acetate (2:1, v/v) mixture
for metabolite analysis. After extraction overnight at room temperature under shaking, the suspension was centrifuged at 21,000 g for
10 min, the supernatant was carefully removed and stored at –20 °C,
and the remaining tissue was extracted overnight with 1 ml of solvent mix as above. Combined extracts were dried under a stream of
nitrogen, and the dry residue was dissolved in 500 µl of ethyl acetate.
An aliquot of 200 µl was again dried under nitrogen, the residue
was dissolved in 100 µl of methanol, and 2–5 µl of this solution was
injected for liquid chromatography–mass spectrometry (LC-MS)
analysis.
Young leaves (~2 cm in length) were chosen for peltate glandular
trichome secretory cell cluster (gland) isolation either for metabolite
extraction or RNA isolation (Gang et al., 2001), and a total of 8 g
of fresh young leaves were used per isolation batch. The S. divinorum
peltate glands were washed and collected from a 33-µm mesh cloth,
yielding about 50 µl of glands per batch. Isolated glands were disrupted by sonication (three 10-s pulses in a Branson 450 sonifer with
a microtip; Branson Ultrasonics) and 1-ml MTBE:ethyl acetate (2:1,
v/v) mixture was added immediately after sonication. The procedure
for metabolite extraction was the same as above.
Metabolite and enzyme assay analyses
Metabolites and products of all enzymatic assays were analyzed
using an LC-MS system (Acquity ultra-performance liquid chromatography coupled to a Synapt G2-S HDMS quadrupole-ion mobility spectrometry–time of flight mass spectrometer; Waters). Extracts
from Salvia tissues were separated on an Acquity UPLC BEH C18
column (50 × 2.1 mm, 1.7 µm; Waters) at a flow rate of 0.45 ml
min–1 using a linear gradient of acetonitrile (B) and water (A) with
0.1% (v/v) formic acid: 0 min, 7% B; 8 min, 99% B; 10 min, 99% B;
10.1 min, 7% B; 13 min, 7% B. Dephosphorylated enzymatic products were separated on a CORTECS UPLC C18+ column (100 × 2.1
mm, 1.6 µm; Waters) using the same gradient and flow rate. Positivemode electrospray ionization (ESI) was applied for ionization. The
Q-TOF-MS instrument parameters were: source 3.2 kV at 100 °C;
desolvation temperature of 250 °C; desolvation gas flow of 800 l h–1.
The transfer collision energy for MSe and MS/MS fragmentation
was 15 eV. Standard curves of serial dilutions of authentic standards
were used for quantification. The direct SdKPS enzyme product and
GGPP were separated on an Acquity UPLC BEH C18 column (50
× 2.1 mm, 1.7 µm; Waters) with 10 mM ammonium bicarbonate
buffer (A) and acetonitrile (B) as the mobile phase using the same
linear gradient and flow rate as above, and were ionized under negative ESI mode.
Cloning and heterologous expression of full-length and
truncated SdKPS and its mutants
The open reading frames of full-length (Fl) and truncated (Tr)
SdKPS were obtained with primers that amplified cDNA from the
targeted sites (SdKPS-FULL-F and SdKPS-exp-R for Fl-SdKPS,
SdKPS-truncation-F and SdKPS-exp-R for Tr-SdKPS; for details
see Supplementary Table S1 at JXB online), followed by transfer
into the pCR2.1 TOPO TA cloning vector (Invitrogen), and verification by complete sequencing. The resulting clones were subsequently transferred to the pEXP-5-CT/TOPO vector by PCR, giving
rise to pEXP/Fl-SdKPS and pEXP/Tr-SdKPS vectors, followed by
sequence verification. Site-directed mutagenesis of Tr-SdKPS was
performed using the QuikChange Lightning kit (Agilent) with the
pEXP/Tr-SdKPS vector. The resulting mutant genes were verified by
1112 | Chen et al.
complete sequencing. The truncated coding sequences of SdKSL1SdKSL3 were cloned into the pCR2.1 TOPO TA vector with restriction enzyme sites, and digested with corresponding restriction
enzymes. SdKSL1 and SdKSL3 were subcloned into the pET15b vector (Merck), and SdKSL2 was subcloned into the pCOLADuetTM-1
(Novagen) expression plasmid. The expression of recombinant proteins was carried out in BL21 StarTM (DE3) (ThermoFisher) cells
induced via the auto-induction method (Studier, 2005) at 18 °C
to 20 °C. Pelleted cells were suspended in binding buffer (20 mM
HEPES, pH 7.5, 500 mM NaCl, 5 mM imidazole, and 5% glycerol)
containing 0.01% (w/v) lysozyme and then disrupted by sonication. The supernatant after centrifugation (20 000 g, 4 °C, 10 min)
was loaded to a pre-equilibrated nickel-nitrilotriacetic acid agarose
matrix (Qiagen). The slurry was incubated with mild agitation at
4 °C for 1 h and then was washed with 10 volumes of washing buffer
(20 mM imidazole). The purified protein was eluted from the resin
with buffer containing 350 mM imidazole and then transferred into
storage buffer (50 mM HEPES, pH 7.5, 1 mM dithiothreitol, and
10% glycerol), and stored at –80 °C if not used immediately.
Enzyme activity characterization
The reaction conditions were optimized in order to achieve reaction
velocity proportional to reaction time and protein amount. Kinetic
parameters were derived from fitting the observed data to the
Michaelis–Menten model (GraphPad Prism 6). The pH optimum
was determined over a pH range from 5.8 to 8.2 in 0.2 unit increments
in 50 mM of Bis-Tris buffer and Bis-Tris-Propane buffer. The concentration of Mg2+ was compared at 0.01 mM, 0.1 mM, 1 mM, and
10 mM. TES, Bis-Tris, HEPES, and Tris at pH 7.0 were compared
as buffer systems. For kinetics measurements, SdKPS enzyme assays
were performed at 30 °C in 250 µl of assay buffer (50 mM HEPES,
pH 7.0, 100 mM KCl, 0.1 mM MgCl2, 10% glycerol). SdKPS activity
was characterized by assaying 0.5 µg purified recombinant protein
incubated with 1–25 µM GGPP in assay buffer for 30 s. Reactions
were terminated as described previously by Prisic and Peters (2007):
a volume of 50 µl 20 mM N-ethylmaleimide (NEM) in 500 mM Gly,
pH 11 was added, followed by incubation at 75 °C for 5 min. Excess
NEM was deactivated by adding 20 µl of 1 M DTT and incubating
for 15 min at room temperature, then neutralizing with 25 µl of 1 M
HCl. Ten units of calf intestine phosphatase (NEB) were added to
dephosphorylate both substrate and product for 30 min at 37 °C.
Catalytic turnover was determined from the ratio of product to the
sum of product and substrate and the known starting GGPP concentration. Kinetic parameters were calculated from three independent assay series, each with two technical replicates. Control assays
included empty vector controls, no-substrate assays, and assays terminated by adding 50 µl of 20 mM NEM and incubation at 75 °C
for 5 min before initiating reaction.
T-DNA construction and transient expression of SdKPS in
N. benthamiana
Agrobacterium tumefaciens strain GV3101:pMP90 was transformed with full-length SdKPS coding sequence in the T-DNA
vector pEarleygate 100 (Earley et al., 2006) by electroporation. The
Agrobacterium infiltration was adapted from previous methods
(Voinnet et al., 2003; Sparkes et al., 2006). Transformed single colonies grown on Luria-Bertani (LB) agar plates containing kanamycin
(50 µg ml–1) and rifampicin (25 µg ml–1) were used to inoculate 2 ml
LB medium plus appropriate antibiotics at 28 °C and 180 rpm for
about 24 h. The pelleted cells (1000 g, 24 °C, 10 min) were washed
with sterile water before being re-suspended in infiltration medium
(27 mM glucose, 50 mM MES, 10 mM MgCl2, 0.1 mM acetosyringone). When several constructs were co-infiltrated, the corresponding Agrobacterium cells were mixed together in equal proportion. The
mixture was infiltrated into the leaves of 4-week-old N. benthamiana
plants. The plants were kept under normal growth conditions for
4 d until metabolite extraction. A. tumefaciens strain GV3101:pMP90
carrying a T-DNA expressing the silencing suppression p19 protein
was mixed together with the construct of interest to suppress RNA
silencing.
Total RNA extraction
For cloning and Illumina cDNA library construction, isolated
glandular trichomes were disrupted by sonication as described
above and RNA was purified using the RNeasy Plant Mini Kit
(Qiagen). Total RNA for quantitative real-time PCR was extracted
from ground leaves of different developmental stages using the
same kit. Genomic DNA was removed using the TURBO DNase
kit (Ambion). Final RNA concentration was determined using a
Nanodrop 2000 (Thermo).
Quantitative real-time PCR
The relative quantification method (Schmittgen and Livak, 2008)
was performed to measure the abundance of SdKPS transcripts.
After DNase treatment, 300 ng total RNA was reverse transcribed into cDNA using qScript™ cDNA Supermix kit (Quanta
BioSciences). Elongation factor-1 (EF-1) was tested with cDNAs
from leaves of different developmental stages and was chosen as
the reference gene for normalization (see Supplementary Table S2).
Primer sequences are shown in Supplementary Table S1 (SdKPS-F
and SdKPS-R, EF-1-F and EF-1-R). Annealing temperatures
between 53 °C and 59.5 °C were compared, and 55 °C was suited
for both SdKPS and SdEF-1. Amplification efficiencies of both
genes were compared using five serial cDNA dilutions. PerfeCta®
SYBR® Green FastMix® (Quanta BioSciences) was used for the
PCR reaction of 95 °C for 2 min, 40 cycles of 95 °C for 10 s, 55 °C
for 15 s, and 70 °C for 20 s, with a subsequent melting curve from
60 °C to 95 °C over 20 min (in a realplex2 Mastercycler EP gradient
S, Eppendorf). cDNA corresponding to 25 ng total RNA was used
as template. Expression levels of SdKPS were normalized to those
of SdEF-1 using the comparative Ct method. Five biological replicates of each leaf pair were analyzed with three technical replicates.
Illumina sequencing and assembly
Total RNAs from peltate trichomes were used for constructing
Illumina paired-end cDNA libraries, sequencing, and read assembly
as described previously by He et al. (2012).
Sequence similarity analysis of S. divinorum diTPSs
Protein sequences were aligned using ClustalX2 (Larkin et al.,
2007). The neighbor-joining algorithm (Saitou and Nei, 1987) was
used with 1000 bootstrap replicates (Felsenstein, 2010) in MEGA6
(Tamura et al., 2013) to generate similarity relationships of candidate S. divinorum diTPSs and other diTPSs. The ent-kaurene/kaurenol synthase from Physcomitrella patens was used to root the tree.
Accession numbers of included proteins are given in Supplementary
Table S3.
Protein structure modeling
Homology models were built using I-TASSER (Zhang, 2008; Roy
et al., 2010) (http://zhanglab.ccmb.med.umich.edu/I-TASSER/). The
model with the highest c-score was selected for structural studies.
Substrate analog AG8 {S-[(2E,6E,10E)-14-(dimethylamino)-3,7,11trimethyltetradeca-2,6,10-trien-1-yl] trihydrogen thiodiphosphate}
was docked with each model as the ligand. The 3D structure of the
labda-13E-en-8-yl+ diphosphate ligand was generated by CORINA
classic (Sadowski et al., 1994) (http://www.molecular-networks.
com). Molecular docking of the ligand into active sites was performed by AutoDock Vina (Steffen et al., 2010). Models and docking results were viewed and manipulated using PYMOL (DeLano
Scientific, South San Francisco, CA, USA).
A diterpenoid synthase involved in salvinorin A biosynthesis | 1113
MALDI-FTICR-MS imaging
Fresh leaves of S. divinorum were affixed to a glass slide using
double-sided tape (3M) and 2,5-dihydroxy benzoic acid (matrix
compound) was applied at a rate of 0.8 mg min–1 (total of 1.07 mg
cm–2 applied) from a solution of 10 mg ml–1 (in methanol–water,
1:1, v/v) by a robotic TM-sprayer (HTX Technologies, Carrboro,
NC). MALDI-MS imaging was carried out using a MALDI solariX
9.4T FTICR mass spectrometer (Bruker Daltonics). The acquisition
mass range was set from 100 to 2000 m/z (mass to charge ratio) with
data obtained in the positive ion mode at 2 Hz and mass resolution
of 66 000 (at m/z 400). The data were obtained at 20 μm spatial resolution. The raw data were then processed and ion maps visualized in
flexImaging 4.1 (Bruker Daltonics).
Accession numbers
Nucleotide sequences of enzymes reported in this study have been
deposited in GenBank with the following accession numbers:
SdKPS, KX268505; SdCPSL1, KX268506; SdCPSL2, KX984339;
SdCPSL3, KX984340; SdCPSL4, KX984341; SdKSL1, KX268507;
SdKSL2, KX268508; SdKSL3, KX268509 and SdEF-1, KX268510.
Results
(–)-Kolavenol and salvinorin A accumulate in peltate
glands of S. divinorum
Our first step in understanding the biosynthesis of salvinorin
A and related diterpenes focused on identifying the tissues
and cellular localization where those diterpenes accumulated.
A previous study indicated that salvinorins are stored in the
subcuticular space of peltate glandular trichomes of S. divinorum, which are located on the abaxial surface of leaves and
stems (Siebert, 2004). They are also present on veins (Fig. 2).
We then analyzed diterpenoids in extracts from young leaves,
stems, and roots. The latter have not been previously tested
for the presence of salvinorin diterpenes, even though the
roots of some Salvia species produce copious amounts of diterpenes (Michavila et al., 1986; Simoes et al., 1986; Ulubelen
et al., 1988; Kolak et al., 2009). (–)-Kolavenol, hardwickiic
acid, and salvinorin A accumulated at comparable levels in leaves and stems, where peltate trichomes are located
(Table 1); however, none of these diterpenoids was detected in
extracts from roots, which lack peltate trichomes. These findings suggest that (–)-kolavenol, hardwickiic acid, and salvinorin A might be produced and/or stored in peltate glands.
Using glass bead abrasion (Gang et al., 2001), peltate
glands were collected from leaves to investigate the diterpenoid content in trichomes directly. As expected, (–)-kolavenol, hardwickiic acid, and salvinorins A and B were readily
detected in the isolated glands. The significantly higher concentrations of the diterpenoids in trichomes compared to
those of whole leaves (Table 2) imply that they are strongly
enriched in peltate trichomes, as has been observed for other
trichome-specific compounds (Berim et al., 2012; Lange and
Turner, 2013).
In an orthogonal direct approach, in situ distribution of salvinorin A-related diterpenoids on the abaxial surface of young
leaves of S. divinorum (2–3 cm in length) was investigated using
MALDI-based imaging mass spectrometry (MALDI-IMS).
Signals corresponding to salvinorin A (471.1421 m/z, [M+K]+)
Fig. 2. Micrographs of peltate trichomes on the abaxial surface of young
leaves of S. divinorum (2–3 cm in length) taken with a light microscope (A,
B) or by SEM (C, D). (A, C) Leaf surface between veins; (B, D) the central
vein. The scale bars are 50 µm in (A, B) and 100 µM in (C, D).
and salvinorin B (429.1316 m/z, [M+K]+), viewed with a mass
window of ±0.1 ppm, appeared as discrete spots distributed on
veins and the leaf blade surface (Fig. 3A, B), which is in line
with the distribution of S. divinorum peltate trichomes (Fig. 2).
In addition, these mass signals overlap almost perfectly
(Fig. 3C). The co-localization pattern of salvinorin A and B
is consistent with the fact that salvinorin B is the proposed
immediate precursor of salvinorin A (Fig. 1B). In contrast,
numerous non-salvinorin pathway-related compounds showed
uniform distribution over the leaf surface (e.g. a randomly
selected signal with m/z 422.9304 is displayed in Fig. 3D) and
did not co-localize with salvinorins A and B (Fig. 3E, F). This
suggested that salvinorins A and B are only localized to specific sites on the leaf surface (glandular trichomes) and that
the other compound was not present in those trichomes. Other
intermediates of salvinorin A biosynthesis (salvinorins, divinatorins, hardwickiic acid, etc.) were also detected and largely
co-localized with salvinorins A and B (Fig. 3).
1114 | Chen et al.
Isolation of a candidate diTPS from S. divinorum
trichome transcriptome assembly
Candidate diTPS genes for involvement in salvinorin A biosynthesis were identified by searching our transcriptome
assembly, generated using RNA from isolated S. divinorum
trichomes, using CPS from Arabidopsis thaliana (AtCPS,
accession number Q38802) as a BLAST query. The bestmatch sequence was subsequently used as the query for additional BLAST searches against the transcriptome assembly.
This exhaustive search revealed a small family of eight putative diterpene synthases belonging to either class I or class II
as defined above. Four of the sequences in the database represented full-length transcripts. Each of these was also used
to further search the database, but no additional related
diTPSs were found. Among the eight genes, a full-length
cDNA named SdKPS (Fig. 4) stood out as having the highest read number per length unit (see Supplementary Table
S4). Phylogenetically, SdKPS clusters with other angiosperm
CPS proteins that fall into the previously defined TPS-c clade,
which contains only the ‘DXDD’ motif (Bohlmann et al.,
1998; Chen et al., 2011). SdKPS is most closely related to
two class II Lamiaceae diTPSs involved in specialized diterpenoid metabolism (Li et al., 2012; Cui et al., 2015) (Fig. 4).
Four incomplete sequences, SdCPSL1–4 (Supplementary
Tables S4, S5; Supplementary Fig. S1), grouped together with
class II diTPSs that produce diterpenoids of ‘normal’ stereochemical configuration (Gao et al., 2009; Caniard et al., 2012;
Sallaud et al., 2012; Schalk et al., 2012; Pateraki et al., 2014;
Zerbe et al., 2014; Cui et al., 2015) (Fig. 4). The other three
proteins, SdKSL1–SdKSL3, which were each full length in
the database, belong to a distinct group of the class I kaurene
synthase-like (KSL) proteins of the TPS-e/f subfamily and
possess the ‘DDXXD’ motif (Bohlmann et al., 1998; Chen
et al., 2011) (Fig. 4). Because of the structure and absolute
configuration of (–)-kolavenol and the fact that salvinorin
diterpenoids are the most abundant diterpenoids in trichomes
Table 1. Accumulation of selected diterpenoids in different tissues
of S. divinorum
Tissue
Leaf
Stem
Root
Concentration (µg g–1 fresh weight)
(–)-Kolavenol
Hardwickiic acid
Salvinorin A
0.23 ± 0.02
0.21 ± 0.04
–
1.7 ± 0.17
1.0 ± 0.18
–
27 ± 1.6
46 ± 11
–
Results are means of four biological replicates ±SE. –, Not detectable.
(Supplementary Fig. S2), the major class II diTPS contig,
SdKPS, was deemed to be more likely to be involved in the
biosynthesis of (–)-kolavenyl diphosphate and was chosen to
be studied in detail.
In vitro enzymatic activity of SdKPS
The open reading frame of SdKPS was directly amplified
from S. divinorum glandular trichome cDNA. Full-length
(Fl) SdKPS transcript appeared to encode a protein with a
putative N-terminal transit peptide (Supplementary Fig. S3,
46 amino acids from the start codon). The truncated (Tr)
recombinant SdKPS (putative transit peptide removed) was
produced in E. coli and purified to near homogeneity using
the C-terminally fused hexahistidine tag.
In vitro assays were carried out with GGPP as the substrate
and the products were analyzed by LC-MS to determine the
enzymatic activity of Tr-SdKPS. The formation of a reaction product (Fig. 5A, peak b in trace 3) was detected in full
assays, but not in controls (Fig. 5A, trace 1 and 2). The mass
to charge ratio (m/z) of the corresponding singly charged
deprotonated molecular ion species [M-H]- was 449.1864
(see Supplementary Fig. S4B), which is consistent with the
formula C20H35O7P2– with a <0.1 ppm error, indicating that
the product of Tr-SdKPS has the same elemental composition as GGPP (Supplementary Fig. S4). This is expected of
(–)-KPP, which is formed by rearrangement and folding of
GGPP. The dephosphorylated product of Tr-SdKPS (peak
c in Fig. 5B, trace 2) was produced using non-specific calf
intestinal phosphatase and identified as (–)-kolavenol based
on its retention time, m/z value, and mass fragmentation pattern, which match perfectly those of the authentic standard
(Fig. 5E). Kinetic analysis of Tr-SdKPS under optimized
assay conditions (Supplementary Fig. S5) revealed that the
reaction followed the Michaelis–Menten model. The determined KM of 1.9 ± 0.66 µM and kcat of 0.88 ± 0.11 s-1, with
kcat/KM of 4.7 × 105 s–1 M–1 (Supplementary Fig. S6) are comparable to those of other characterized plant diTPSs with
their native substrates (Prisic and Peters, 2007; Keeling et al.,
2008). Taken together, the in vitro assay results suggested that
SdKPS acts as the monofunctional (–)-kolavenyl diphosphate
synthase (KPS) initiating the biosynthesis of (–)-kolavenol in
S. divinorum.
We also sought to elucidate the enzyme responsible for
dephosphorylation of (–)-KPP in S. divinorum by expressing recombinant SdKSL1–3 for biochemical assessment.
Both SdKSL1 and SdKSL3 were successfully expressed with
their N-terminal transit peptides removed and were subsequently purified (see Supplementary Fig. S7). However, the
Table 2. Accumulation of selected diterpenoids in whole leaves and peltate glandular trichomes of S. divinorum. Results are means of
four biological replicates ±SE
Tissue
Leaf
Gland
Concentration (µg g–1 fresh weight of tissue)
(–)-Kolavenol
Hardwickiic acid
Salvinorin B
Salvinorin A
0.200 ± 0.031
1100 ± 170
2.00 ± 0.27
15 000 ± 1100
31.0 ± 1.6
150 000 ± 24 000
30.0 ± 2.3
250 000 ± 13 000
A diterpenoid synthase involved in salvinorin A biosynthesis | 1115
Fig. 3. MALDI-FTICR-MS imaging of diterpenoids (including salvinorin A and salvinorin B) on the abaxial surface of a S. divinorum leaf. The data were
obtained at 20 μm spatial resolution and a mass resolution of >58 000 for all compounds measured (66 000 at m/z 400), all from the same leaf. Scale
bars = 1 mm. Each panel displays the distribution of the mass signal for a specific compound or pairs of compounds using a ±0.1 ppm mass window
(achievable using the Bruker MALDI-SolariX 9.4T FTICR instrument) around the monoisotopic mass for each compound. (A) Salvinorin A ([M+K]+, m/z
471.1421) and (B) salvinorin B ([M+K]+, m/z 429.1316). (C) The merged image of (A) and (B). (D) Distribution of an abundant, non-salvinorin-related
compound (m/z 422.9304). (E) The merged image of (A) and (D). (F) The merged image of (A), (B), and (D). (G) Hardwickiic acid ([M+K]+, m/z 355.1676),
(H) divinatorin A ([M+K]+, m/z 371.1625), (I) divinatorin B ([M+K]+, m/z 401.1730), (J) divinatorin E ([M+K]+, m/z 399.1574), (K) divinatorin F ([M+K]+, m/z
417.1680) and (L) salvinorin F ([M+K]+, m/z 413.1367).
co-incubation of SdKPS with either SdKSL1 or SdKSL3 and
with GGPP as substrate did not result in any dephosphorylated product being formed. SdKSL2 was not expressed in
E. coli to any detectable level under the same expression conditions as were used for SdKSL1 and SdKSL3, and the enzyme
activity was no higher than the background. Therefore, the
enzyme catalyzing the conversion of (–)-KPP into (–)-kolavenol in S. divinorum remains unknown.
diphosphate can be dephosphorylated to (–)-kolavenol by an
endogenous enzyme from N. benthamiana leaves. It has been
previously suggested that endogenous phosphatases can convert the product of class II diTPSs into corresponding diterpenols in tobacco (Sallaud et al., 2012; Zerbe et al., 2014,
2015). Thus, in vivo functional characterization of Fl-SdKPS
confirmed the results obtained in vitro with Tr-SdKPS.
Transient expression of SdKPS in planta
Developmental pattern of diterpenoid accumulation
and SdKPS expression
S. divinorum is currently not amenable to genetic transformation. Therefore, the function of our candidate KPS was
tested by infiltrating the leaves of N. benthamiana with
A. tumefaciens strains carrying Fl-SdKPS. Protein p19 was
co-expressed with Fl-SdKPS to improve the expression of
the transgene (Voinnet et al., 2003). (–)-Kolavenol could only
be detected in extracts from leaves where the expression of
Fl-SdKPS was expected to occur (Fig. 5C, trace 3; Fig. 5E),
while it was not present in either untreated controls or in leaves
infiltrated only with the p19 enhancer strain (Fig. 5C, trace 1
and 2), suggesting that Fl-SdKPS was successfully expressed
and the encoded protein was catalytically active in N. benthamiana leaves. These results not only confirm the function
of Fl-SdKPS as KPS, but also indicate that (–)-kolavenyl
The diterpenoid synthase required for (–)-kolavenol production is the first enzyme of the salvinorin A pathway (Fig. 1B).
Therefore, analysis of relationships between accumulated diterpenes and SdKPS gene expression might further confirm or
refute whether SdKPS plays the proposed role in planta. For
this experiment, whole leaves of three developmental stages
from five plants of the same age were used. Leaf pairs with
distinct size and age were only collected from the main stem,
with 1st pairs being the youngest (from the top node), the 3rd
pairs being the middle (from the 3rd node), and the 5th pairs
(from the 5th node) being the oldest at time of collection.
The salvinorin-related diterpenoids evaluated were
(–)-kolavenol, hardwickiic acid, salvinorin B, and salvinorin A. (–)-Kolavenol and hardwickiic acid were monitored
1116 | Chen et al.
monitored developmental time course. Salvinorins A and B
accumulated at much higher levels. The concentrations of
both compounds decreased abruptly in the 3rd leaf pairs
compared to the 1st, and then leveled off (Fig. 6B). In parallel
with metabolite levels, the expression of SdKPS was highest
in young leaves and decreased dramatically as the leaves aged.
The difference of expression was 4-fold between the 1st and
3rd pair as well as between the 3rd and 5th pair (Fig. 6A).
Although the transcript abundance may not reflect the enzymatic activity in the glands, the relationship of the differences
between metabolite profiles and expression of SdKPS is in
line with its proposed physiological role in S. divinorum.
The basis for the product specificity of SdKPS
Fig. 4. The phylogenetic relationship of S. divinorum diTPSs and other
published class I and class II diTPSs, as inferred using the NeighborJoining method. Bootstrap values (1000 replicates, shown if >70%) are
given next to the branches. The scale represents the number of amino
acid substitutions per site. The ent-kaurene/kaurenol synthase from
Physcomitrella patens was used to root the tree. For the convenience of
phylogeny analysis, we generated the putative chimeric protein sequence,
named as SdCPSL234, by combining the translated sequences of
SdCPSL2, SdCPSL3, and SdCPSL4 (see Supplementary Fig. S1), as
they are likely to be the same gene (Supplementary Tables S1 and S2).
Descriptions of proteins are provided in the Materials and Methods and
Supplementary Table S4.
because they are closely connected to the KPS-catalyzed
reaction and are early intermediates in the pathway, while
salvinorin A and B are the principal products of the pathway (Kutrzeba et al., 2009a) and thus represent the overall
flow through the metabolic pathway. For all four diterpenoids
analyzed, the levels of individual compounds were highest in
the youngest leaf pair and decreased as the leaves matured
(Fig. 6), following one common pattern of biosynthesis in
Lamiaceae peltate glandular trichomes. (–)-Kolavenol, the
earliest detectable intermediate of salvinorin A biosynthesis,
accumulated at lower concentrations (0.03–0.23 µg g–1) than
the other three diterpenoids, potentially reflecting its efficient
conversion into compounds downstream in the pathway.
The concentrations of hardwickiic acid were slightly higher
than those of (–)-kolavenol (0.25–1.66 µg g–1) through the
Although SdKPS is phylogenetically closely related to several
characterized plant CPSs (Fig. 4), it exhibits a distinct function. Of the currently known CPSs, SdKPS shares the highest
protein identity of 72% with IeCPS2 from Isodon eriocalyx
(Li et al., 2012). The differences between these proteins are
sufficient to direct IeCPS2 to deprotonate the labda-13Een-8-yl+ diphosphate at C-17, whereas SdKPS first proceeds
through a series of 1,2-hydride and methyl shifts, and concludes with a deprotonation at C-3, resulting in (–)-KPP
(Fig. 1A). A comparison of these two proteins appeared,
therefore, to be a productive approach to identify the residues of SdKPS that mediate its product specificity. The predicted protein structure of SdKPS was first aligned with that
of IeCPS2, and divergent residues within 4- and 8-Å spheres
around C-8 of the substrate analogue in the structure of CPS
from Arabidopsis thaliana were identified (Protein Data Bank:
3PYA) (Köksal et al., 2011, 2014) and superimposed on our
models by I-TASSER (Zhang, 2008). The sequence of SdKPS
was then aligned with those of other related, functionally
characterized plant CPSs (see Supplementary Fig. S3) to find
residues unique to the active site of SdKPS (Table 3) and thus
most likely to be involved in determining its product. In order
to assess the effect of those residues on enzymatic function
of SdKPS, they were replaced by the corresponding residues
found in IeCPS2 by site-directed mutagenesis. The activities
of the purified mutant proteins were then tested in vitro.
Three divergent residues, V200, F255, and A314 were identified within the predicted 4-Å sphere in SdKPS, corresponding to I200, H265, and V315 in IeCPS2. Most notably, the
SdKPS:F255H mutant showed a complete switch of product
specificity from (–)-KPP to ent-CPP (Fig. 5D). The other single mutations, V200I or A314V, on the other hand, did not
change the product outcome (see Supplementary Fig. S8).
Intriguingly, the double-mutant, A314V/F255H, led to the
recovery of the ability to produce (–)-KPP while retaining
the acquired function of forming ent-CPP (Fig. 5D). None
of the divergent residues within the 4–10-Å sphere as shown
in Table 3 redirected the catalytic routes from route a to
route b (Fig. 1A), as revealed by product profiles of mutants
SdKPS S369V, SdKPS S369V/S372T/S373A, and SdKPS
S402C (Supplementary Fig. S8). Thus, it appears that only
two residues modulate the product outcome of SdKPS relative to CPS.
A diterpenoid synthase involved in salvinorin A biosynthesis | 1117
Fig. 5. Functional characterization of SdKPS and relevant SdKPS mutants. (A) Extracted ion chromatogram (EIC) from LC-MS analysis under negative
ionization mode of the non-dephosphorylated reaction product (peak b) of recombinant Tr-SdKPS using GGPP (peak a) as substrate (trace 3), compared
with the reaction where there is no Tr-SdKPS (trace 1) or deactivated Tr-SdKPS (trace 2). The reactions are indicated at the upper-right corner of each
panel. (B) EIC from LC-MS analysis under positive ionization mode of the dephosphorylated reaction product of recombinant Tr-SdKPS using GGPP
as substrate (trace 2), compared with non-dephosphorylated reaction product (trace 1) and authentic standard of (–)-kolavenol (trace 3). Peak c,
(–)-kolavenol; peak d, geranylgeraniol (GGOH, dephosphorylated GGPP). (C) Transient expression of Fl-SdKPS in leaves of Nicotiana benthamiana. EIC of
metabolite extracts from N. benthamiana leaves (trace 1) or leaves after Agrobacterium-mediated transient co-expression of Fl-SdKPS with p19 for RNAsilencing suppression (trace 3) or leaves expressing p19 alone (trace 2) analyzed under the same LC-MS conditions applied in (B). (D) Effects of F255H
and F255H/A314V mutations on the SdKPS product profile. EIC (m/z 273) from LC-MS analyses of the dephosphorylated products of reactions indicated
in each panel. A different UPLC column from that in (A–C) was used, as detailed in the Materials and Methods. Peak e, ent-copalol. (E) Top panel: MS/
MS spectrum of peak c produced by Tr-SdKPS after dephosphorylation as in Fig. 5B (trace 2), compared to that of authentic (–)-kolavenol. Bottom panel:
comparison of MS/MS spectra of peak c detected in extract of Fl-SdKPS-transformed N. benthamiana leaves in Fig. 5C (trace 3) and that of authentic
(–)-kolavenol.
Discussion
Biosynthesis of (–)-kolavenol in trichomes of
S. divinorum
SdKPS was identified as a mono-product class II diTPS
catalyzing the formation of (–)-KPP from GGPP as the
first reaction in salvinorin A biosynthesis (Figs 1 and 5).
Formation of (–)-KPP presumably starts from the protonation-initiated cyclization of GGPP, proceeds through
a series of 1,2-hydride/methyl migrations on the initial
intermediate labda-13-en-8-yl+ diphosphate, and is terminated by deprotonation (Fig. 1A, route a). The subsequent
dephosphorylation of (–)-KPP could be catalyzed by a phosphatase or by a class I diTPS, a class of enzymes that mediates the formation of other labdane-related diterpenoids
with or without subsequent hydroxylation of the dephosphorylated product (Zi et al., 2014), including those with
the clerodane backbone (Andersen-Ranberg et al., 2016; Jia
et al., 2016).
As salvinorin A biosynthesis and storage primarily occurs
in peltate trichomes in S. divinorum (Table 2; Figs 2 and 3), the
concentration of diterpenoids in whole-leaf tissue is mainly
determined by biosynthesis rates in individual trichomes and
by trichome density. It is possible that the de novo biosynthesis
1118 | Chen et al.
of diterpenoids slows down or nearly ceases as the trichomes
mature, as observed for monoterpenoid production in peppermint (McConkey et al., 2000; Turner et al., 2000).
Fig. 6. Accumulation of selected diterpenoids and transcript levels of
SdKPS in S. divinorum leaves of different growth stages. (A) Comparison of
(–)-kolavenol accumulation and SdKPS expression in the same leaf tissue
of different growth stages. (B) Content of hardwickiic acid, salvinorin B,
and salvinorin A in the same leaf tissue as in (A). Different letters indicate
statistically significant differences in the metabolite concentrations in
different leaf pairs as calculated using a one-way ANOVA test (P<0.05) with
a Tukey HSD test. Results are the means of five biological replicates ±SE.
Identification of a highly specific enzyme catalyzing the
formation of (–)-KPP
Several studies have demonstrated extreme product plasticity of class II (Criswell et al., 2012; Potter et al., 2014, 2015,
2016; Mafu et al., 2015; Jia et al., 2016) and class I diTPSs
(Wilderman and Peters, 2007; Xu et al., 2007b; Keeling et al.,
2008; Morrone et al., 2008; Zerbe et al., 2012; Irmisch et al.,
2015), providing evidence for the crucial role of neo-functionalization in the rise of diterpenoid chemical diversity. A very
recent structure–function investigation of the catalytic histidine in AtCPS found that its substitution for the aromatic
residues tyrosine or phenylalanine causes the enzyme to produce two novel products, one of them (–)-KPP (Potter et al.,
2015). Moreover, the reciprocal mutation of phenylalanine to
histidine in SdKPS (F255H), as demonstrated herein, completely converts its enzymatic activity to CPS.
The substitution with a histidine of F255 not only forms a
catalytic base group present in plant CPSs (Potter et al., 2014)
for deprotonation at C-17 of labda-13E-en-8-yl+ diphosphate
intermediate, the orientation and location of the intermediate in the active site was also altered, as revealed by molecular docking (Fig. 7). The distance from C-17 of the ligand
to F255 in SdKPS is longer than that from C-17 to H255 in
SdKPS:F255H (Fig. 7A, B). In addition, the substitution of
alanine with valine at residue 314 partially restores the KPS
activity of SdKPS:F255H, giving rise to production of both
(–)-KPP and ent-CPP. Residue 314 is located in close proximity to F255 and N313 in the predicted active-site cavity of
SdKPS, and it lines the surface of the cavity (Fig. 7D). The
larger side chain of valine compared to alanine presumably
leads to an alteration of the steric shape of the side of the
reaction pocket and the positioning of the labda-13E-en8-yl+ diphosphate intermediate. Thus, C-17 of the intermediate is not efficiently deprotonated by H255 in the active-site
pocket of SdKPS:F255H/A314V, which enables hydride and
methyl migrations in the backbone rearrangement that leads
to (–)-KPP formation.
The functional conversions of SdKPS by F255H mutation
and AtCPS by H263F (Potter et al., 2016) also pinpoint the
catalytic histidine as a critical structural element of class II
diTPSs underlying diversification of labdane-related diterpenoid biosynthesis in plants. The histidine in plant CPSs
belongs to the catalytic His-Asn dyad of CPSs (H255 and
Table 3. Divergent residues in SdKPS versus IeCPS2 models within 8 Å of the central carbon atom of the substrate analog in the
structure of ent-CPS from A. thaliana and the corresponding residues in other plant ent-CPSs
Radius, Å
4
4
4
8
8
8
10
Residue in
SdKPS
IeCPS2
PsCPS
CmCPS2
AtCPS
LsCPS
SrCPS
V200
F255
A314
S369
S372
S373
S402
I200
H265
V315
V370
T373
A374
C403
I
H
V
I
T
A
C
I
H
V
I
T
A
C
I
H
V
I
T
A
C
I
H
V
I
T
A
C
I
H
V
I
T
A
C
A diterpenoid synthase involved in salvinorin A biosynthesis | 1119
Fig. 7. Models of active site cavities of SdKPS and related proteins. Active-site cavities are displayed for SdKPS (A), SdKPS:F255H (B), AtCPS:H263F
(C), and SdKPS:F255H/A314V (D) modeled with labda-13E-en-8-yl+ diphosphate as the ligand. In (A–C) cavities are viewed from the entrance; in (D) the
cavity is viewed from outside of the cavity wall. Carbon atoms in the backbone and surface of cavities are colored purple in (A), yellow in (B), cyan in (C),
and orange in (D) to represent active sites from different enzymes. The grey shading represents the protein structure outside of the cavity side wall.
N313 in SdKPS:F255H) (Potter et al., 2014). This His residue
was conserved in all known plant CPSs producing ent-CPP,
including the bifunctional diterpene cyclase (PpCPSKS) from
the moss Physcomitrella patens, suggesting that it originated
more than 400 million years ago (Hayashi et al., 2006; Potter
et al., 2014; Zi et al., 2014). A divergent residue at this position appears to underlie the neo-functionalization of class II
diTPSs, which resulted in expansion of labdane diterpenoid
diversity in the plant kingdom. This notion is supported by
the presence of aromatic residues (phenylalanine or tyrosine)
at positions corresponding to SdKPS F255 in many other
class II diTPSs involved in specialized biosynthesis of various labdane-type diterpenoids from different angiosperm lineages (see Supplementary Table S6). Specifically, a tyrosine is
found at the corresponding position in the only other identified plant (–)-KPP synthase, TwTPS14 (Andersen-Ranberg
et al., 2016), supporting the idea that a stable aromatic residue at this position is essential for production of (–)-KPP.
Besides F255, there must be other factors contributing to
product specificity of SdKPS, as many other enzymes with
the F/Y residue do not produce (–)-KPP (see Supplementary
Table S6) and AtCPS:H263F produces a secondary product,
ent-labda-13E-en-8α-ol diphosphate. The inability of SdKPS
to produce ent-labda-13E-en-8α-ol diphosphate indicates a
unique strategy for preventing access to water by C-8 of the
intermediate, which AtCPS:H263F lacks (Potter et al., 2015).
Intriguingly, the size and shape of the predicted active-site
cavity of SdKPS differ from those of AtCPS:H263F (Fig. 7A,
C). The labda-13E-en-8-yl+ diphosphate intermediate could
be stabilized in a different orientation, where C-8 is more distant from the residue at position 255 in the cavity of SdKPS
(Fig. 7A, C). Although the crystal structure of SdKPS has
not been solved experimentally, the docking results based on
the predicted SdKPS cavity structure suggested that, along
with the steric block by phenylalanine, the orientation and
position of C-8 of the intermediate, probably determined
by the shape and size of the active-site cavity, enable the full
elimination of hydroxylation on C-8. A high-resolution crystal structure of SdKPS will help answer these questions.
The evolution of kolavenol biosynthesis
SdKPS represents one of the very few examples of a diTPSs
forming the clerodane backbone in plants. The only other
known plant diTPS catalyzing a similar reaction is the very
recently reported class II diTPS TwTPS14 from Tripterygium
wilfordii (Celastraceae) (Andersen-Ranberg et al., 2016). The
low protein sequence identity (53%) and the phylogenetic
relationship between SdKPS and TwTPS14 suggest that
the ability to produce (–)-kolavenyl diphosphate arose independently in these two plant lineages. Phylogenetic analysis (Fig. 4) shows that SdKPS clusters together with other
Lamiaceae diTPSs involved in specialized diterpenoid biosynthesis. On the other hand, TwTPS14 is much more closely
1120 | Chen et al.
related to functionally different diTPSs from T. wilfordii
(Andersen-Ranberg et al., 2016). The convergent or repeated
evolution scenario (Pichersky and Gang, 2000; Pichersky and
Lewinsohn, 2011) of kolavenol biosynthesis is also evident
in the fact that a diTPS catalyzing the formation of (+)-KPP
has recently been isolated from Herpetosiphon aurantiacus, a
filamentous green non-sulfur bacterium producing the clerodane diterpenoid methylkolavelool (Nakano et al., 2015).
While it also contains the DXDD motif and is classified as
a class II diTPS, the overall protein identity of the bacterial
diTPS with SdKPS is less than 25%.
The independent evolution of kolavenol biosynthesis in the
plant kingdom might be driven by the selective pressure provided
by the insect anti-feedant activity of clerodanes. Indeed, the
higher concentration of the clerodane diterpenoids in younger
leaves and insect anti-feedant activities of various clerodanes
(Sosa et al., 1994; Klein Gebbinck et al., 2002; Rosselli et al.,
2004; Sivasubramanian et al., 2013) suggest that they may serve
to protect young susceptible tissues from herbivore attack.
Supplementary data
Supplementary data are available at JXB online.
Table S1. All primers used in this study.
Table S2. The expression of EF-1 in leaf tissues of different
growth stages, presented as 2–CT.
Table S3. List of the names and accession numbers of proteins displayed in Fig. 4.
Table S4. Reads per kilobase of transcript per million
mapped reads for candidate SdCPSL contigs.
Table S5. The top three matches of SdCPSL2-4 in
UniProtKB/Swiss-Prot identified by a BLAST search.
Table S6. Characterized class II diTPSs for specialized biosynthesis of various labdane-type diterpenoids that contain
aromatic residues (phenylalanine or tyrosine) at the same
position as SdKPS F255.
Fig. S1. Sequence alignment of SdCPSL2-4, SdCPSL234,
and homologous CPSLs in Table S5.
Fig. S2. Total ion chromatogram from LC-MS analysis
under positive ionization mode for peltate trichome metabolite extract.
Fig. S3. Sequence alignment of SdKPS and CPSs.
Fig. S4. LC-MS/MS spectra of peaks in Fig. 5.
Fig. S5. Determination of optimal conditions for in vitro
enzymatic reaction of SdKPS.
Fig. S6. A representative hyperbolic plot of SdKPS reaction rate vs. substrate concentration.
Fig. S7. SDS-PAGE of purified recombinant SdKSL1
and SdKSL3 and their putative N-terminal transit peptide
sequences.
Fig. S8. LC-MS extracted ion chromatograms (m/z 273) of
products of SdKPS and other mutants with GGPP.
Acknowledgements
We thank Dr Jeong-Jin Park (Tissue Imaging and Proteomics Laboratory,
Washington State University) for support with MALDI-FTICR-MS analysis
and Mr Robert Long (Washington State University) for cultivation of S. divinorum. We gratefully acknowledge Dr Ying Zeng (State Key Laboratory of
Phytochemistry and Plant Resources in West China, Kunming Institute of
Botany, Chinese Academy of Sciences) for generously providing us with the
pETTR-IeCPS2 plasmid. Financial support for XC was provided by the
Global Plant Sciences Initiative Research Fellowship from the Program of
Molecular Plant Sciences at Washington State University.
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